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Cloning and characterisation of selected Class II formins
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Rösel, Daniel (referee)
Formins are proteins involved in regulation and construction of actin filaments of eucaryotic organism. They parcipitate in regulating cytokinesis, polar tip growth, and thus participate in development of whole organisms. There are 2 classes of formins in Arabidopsis thaliana. Both classes include FH1 and FH2 domains (formin homology 1 a 2). Class I formins have N-terminal transmembrane domain, unlike class II formins. Some formins of class II have a N-terminal PTEN domain (Phosphatase and Tensin Homolog). Sequence analyses predicted membrane binding via phosphatase or C2 subdomain of PTEN. This thesis was focused on the formin AtFH14, specifically its PTEN domain. Based on predicted sequence, a DNA fragment encoding the PTEN domain was amplified, sequenced and cloned to destination vectors for YFP and EOS phusions. Marked protein was visualized by transient expression in Nicotiana benthamiana. Stably transformed Arabidopsis lines were prepared for stably expression of protein. The tagged protein was localized in cortical cytoplasm, cytoplasmatical strands, probably in nuclear membrane or perinuclear cytoplasm, as well as in peculiar "folicle-like" structures that might be due to binding of PTEN at the periphery of some membrane organelles. Also were seen filament structures, maybe caused by PTEN binding...
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The role of actin dynamics in auxin transport
Stillerová, Lenka ; Cvrčková, Fatima (advisor) ; Schwarzerová, Kateřina (referee)
Phytohormones are signalling molecules directing physiological and developmental processes in plants. One of them, auxin, is involved in the diverse regulation of plant processes, e.g. embryogenesis, organogenesis, vascular tissue formation and tropisms. Auxin transport is polar. Auxin isdistributed via the phloem, utilizing specialized membrane transport proteins; small amount diffuse also through the membrane. Aux1/Lax transporters mediate auxin entry into the cell, auxin efflux is mediate mostly by PIN transporters, which are the crucial factors in determining the directionality of auxin flow. Asymmetric localization of membrane PIN proteins depends on vesicle transport from Golgi to the plasma membrane. Vesicles are transported along actin filaments which are dynamically rebuilted by regulators. They are maintaining asymmetric cellular localization of the auxin transport proteins. PIN proteins are cycling between endosomes and plasma membrane. Cycling is regulated by ARF-GEF proteins and serin/threonin kinase (PID, PINOID). Newly synthesized PIN proteins are equally distributed in the plasma membrane, afterwards they are asymmetrically redistributed. Regulation of actin filaments formation and remodelling is the crucial factor for transport of vesicles with PIN proteins. Many proteins which regulate...
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